Various bacterial products have the capability to exert undesirable effects on humans and other animals. Endotoxins are toxic materials released by bacterium on bacterial lysis and are distinguished from the toxic substances synthesized and excreted by the intact bacterium, namely exotoxins. While endotoxins were first recognized for their ability to induce fever, they are now known to have a broad spectrum of biologic activities. On bacteriolysis, endotoxins consisting of aggregates of lipopolysaccharides and protein and to some extent loosely bound lipids, are released from the bacterium into the surrounding medium. Thus, endotoxins consist primarily of lipopolysaccharides (LPS) with various amounts of protein and lipid. Since it has been demonstrated that almost all of the biologic activities usually attributable to bacterial endotoxins can also be elicited with isolated chemically pure lipopolysaccharides, the terms "endotoxins" and "lipopolysaccharides" have been utilized interchangeably to some extent.
Pyrogens, particularly endotoxins or lipopolysaccharides, are constituents of the cell wall of Gram-negative bacteria. When admitted to the circulation of animals, these materials trigger a chain of biochemical reactions which lead to the production of an elevated body temperature or fever; hence, the name pyrogens. Removal of pyrogens from pharmaceutical preparations intended for parenteral use has long been a problem due to the ubiquitous distribution, detergent-like properties and the stability of the endotoxin molecules and the extreme potency of their fever-inducing activity. Current conventional methods for the removal of pyrogens employ either roasting, strong acid or base treatment, or chemical oxidation, none of which is an acceptable treatment for use with sensitive pharmaceutical preparations. Ultrafiltration is an effective method for sensitive preparations, but is useful only for low (less than 10,000 MW) molecular weight preparations. Molecular sieve chromatography has the same limitations as ultra filtration. Thus, there is no current method of general applicability for removal of pyrogens from macromolecular preparations.
In the administration of intravenous fluid, it is imperative that no endotoxins be administered to the patient. Although intravenous fluids are processed so as to eliminate any microorganisms, such as by autoclaving, for example, in intravenous therapy it is common to use a filter at the time of administration in an attempt to remove any microorganism which may remain in the fluid. Such filters are described, for example, in U.S. Pat. Nos. 3,854,907 and 4,101,423. These filters are not effective to remove endotoxins.
A current means of detecting and quantitating endotoxin in vitro is the Limulus Amoebocyte Lysate method (LAL) as described in the New England Journal of Medicine, Vol. 289, No. 18, pages 931 to 934, Nov. 1, 1973. Limulus amoebocyte lysate is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL forms a firm clot when incubated with endotoxins and can be used to detect small quantities of endotoxin. A limulus lysate of improved sensitivity is described in U.S. Pat. No. 4,107,077, granted Aug. 15, 1978. LAL is sold commercially under the trademark Pyrotell by Associates of Cape Cod, Inc. and is used in the detection and quantitation of endotoxin.